The potential advantages of expanded beds, i.e., fluidized beds, of ion exchange or affinity adsorbents for direct adsorption of proteins from biological process liquids and particulate-containing fluids has been demonstrated. Conventional fluidized bed adsorption systems use 100 .mu.m to 400 .mu.m polymeric or composite particles with small density differences between the adsorbent particles and the liquids being processed. The wide particle size distribution of these adsorbents results in stable beds of particles being classified by the fluid velocity and density. Even though these classified expanded beds are stable, many of these particles are large and have a long characteristic diffusion length (i.e., path length from the outer surface of the particle to the center) within the adsorbent. This results in poor adsorption kinetics. Furthermore, such adsorption particles lack robust ligands with high specificity, are unable to be repeatedly cleaned with harsh reagents, and cannot be used for protein adsorption at elevated temperatures (i.e., greater than about 40.degree. C.). In addition, loss of bed capacity with increasing bed expansion (liquid flow rate) using conventional fluidized bed adsorbents may limit fluidized bed separations due to pore mass transfer resistance.
Solutions to these and other problems associated with conventional fluidized bed systems have included the use of magnetically stabilized fluidized beds (MSFB) and the division of the bed into stages. A more useful approach would be to develop stable adsorbent particles of higher density with appropriate adsorption properties. A need exists for such particles. It is envisioned that with denser particles, higher fluidization velocities (100-250 cm/hour) can be achieved with smaller adsorbent particles. While high flow rates can also be achieved using larger particles (200 .mu.m to 500 .mu.m), fluidization of small particles will minimize bed dispersion and result in more rapid protein adsorption in the presence of entrained particulates such as fermentation broths, cell lysates, blood, or cell culture fluids.
Porous ceramic particles, such as silica, as well as highly crosslinked functionalized organic polymeric materials are used in high performance liquid chromatography for the separation of proteins. However, most of these particles do not have the appropriate density for effective use in expanded or fluidized beds. Furthermore, the ability to repeatedly remove adsorbed protein, nucleic acids, lipids, pyrogenic lipopolysaccharides (LPS), and intact virus or microorganisms (bacteria, fungi or yeast) from such chromatographic media is a challenging problem. This makes these materials undesirable for use in fluidized bed applications because process scale protein adsorbents useful for purification of therapeutic or diagnostic proteins must be capable of repeated clean-in-place cycles. Both the adsorbent and the surface need to be stable to cleaning without loss of capacity or mechanical stability. Such cleaning methods are generally harsh: high or low pH solutions (0.1-2 M NaOH, formic, acetic, peracetic, trifluoroacetic, or hydrochloric acid to 1 M) often in alcohol (70% ethanol or 30% isopropanol); high ionic strength solutions (2 M NaCl or KCl); non-ionic detergents; or high temperature conditions followed by extensive washing with purified, sterilized buffer. Particularly vigorous methods, often at elevated temperatures (e.g., 40-80.degree. C.), are needed to remove and inactivate lipopolysaccharide endotoxin (LPS) and viral nucleic acids from protein adsorbent media because of the extremely low residual levels of these contaminants allowed in human biologics as established by federal regulation and the World Health Organization. Some silica-coated and organic polymeric chromatographic adsorbents tolerate cleaning with 0.1 M sodium hydroxide or ethanol-acetic acid mixtures at refrigerated (4.degree. C.) or ambient temperatures and are more mechanically stable than carbohydrate adsorbents. In general, however, organic polymeric adsorbents cannot be cleaned with harsh agents at elevated temperatures (e.g., 40-60.degree. C.) or sterilized with steam or direct heat. Furthermore, silica chromatographic adsorbents typically cannot be rigorously sanitized, for example, with 0.2 M to 1 M sodium hydroxide, without degradation. In general, silica-based materials are not stable outside the pH range of 2 to 8.
Small (&lt;25 .mu.m) surface-modified porous and highly dense zirconium oxide particles are used in high performance liquid chromatography separations of proteins. See, for example, J. A. Blackwell et al., J. Chromatogr., 549, 59 (1991); J. A. Blackwell et al., J. Chromatogr., 596, 27 (1992); P. W. Carr et al., Chromatography in Biotechnology, American Chemical Society, Washington D.C., Symp. Ser. 529, 146 (1993); J. Nawrocki et al., J. Chromatogr. A, 657, 229 (1993); and J. Nawrocki et al., Biotechnol. Prog., 10, 561 (1994). Such surface-modified zirconium oxide particles can be modified with a variety of Lewis bases. Only certain of these surface-modified zirconium oxide particles, however, are capable of withstanding the repeated harsh cleaning conditions required for process scale separation of polypeptides and proteins destined for therapeutic use. Furthermore, such particles do not have the appropriate particle size for effective use in an expanded fluidized bed.
Although larger zirconium oxide particles (1 .mu.m to 1 cm), have been suggested as suitable for use in fluidized beds (see, U.S. Pat. Nos. 5,108,597, 5,271,833, and 5,346,619), these particles are either carbon-clad or carbon-clad with a crosslinked polymer. Such surface-modified zirconium oxide particles are used for reversed phase separations and are not generally suitable for expanded bed and most process scale protein separations. See, for example, C. H. Lochmuller et al., Preparative Chromatography 1, 93 (1988). Furthermore, protein adsorption at elevated temperatures and repeated cleaning of these particles with strong base (0.2 M to 1.5 M NaOH) has not been disclosed. Thus, a need exists for adsorbent particles of the appropriate particle size, density, porosity, and high temperature stability for use in process scale expanded beds for purification of therapeutic or diagnostic proteins.